Compounds and methods for the detection of methotrexate
The disclosure relates generally to methods of detecting and quantifying methotrexate (MTX) in a sample. The methods disclosed herein decrease cross-reactivity and improve sensitivity of the detection.
1. An isolated antibody that binds to methotrexate (MTX), the antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain nucleotide sequence comprises SEQ ID NO:1 and wherein the variable light chain nucleotide sequence comprises SEQ ID NO:2.
2. An isolated antibody that binds to methotrexate (MTX), the antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain nucleotide sequence comprises SEQ ID NO:5 and wherein the variable light chain nucleotide sequence comprises SEQ ID NO:6.
3. An isolated antibody that binds to methotrexate (MTX), the antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain nucleotide sequence comprises SEQ ID NO:9 and wherein the variable light chain nucleotide sequence comprises SEQ ID NO:10.
4. An isolated antibody that binds to methotrexate (MTX), the antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain amino acid sequence comprises SEQ ID NO:3 and wherein the variable light chain amino acid sequence comprises SEQ ID NO:4.
5. An isolated antibody that binds to methotrexate (MTX), the antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain amino acid sequence comprises SEQ ID NO:7 and wherein the variable light chain amino acid sequence comprises SEQ ID NO:8.
6. An isolated antibody that binds to methotrexate (MTX), the antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain amino acid sequence comprises SEQ ID NO:11 and wherein the variable light chain amino acid sequence comprises SEQ ID NO:12.
7. The method of detecting methotrexate (MTX) in a sample, comprising:
combining in a solution the sample with a capture molecule and a labeled MTX, wherein the capture molecule comprises a solid support and an isolated antibody that binds to methotrexate (MTX), the isolated antibody selected from the group consisting of (1) an antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain nucleotide sequence comprises SEQ ID NO:1 and wherein the variable light chain nucleotide sequence comprises SEQ ID NO:2, (2) an antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain nucleotide sequence comprises SEQ ID NO:5 and wherein the variable light chain nucleotide sequence comprises SEQ ID NO:6, (3) an antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain nucleotide sequence comprises SEQ ID NO:9 and wherein the variable light chain nucleotide sequence comprises SEQ ID NO:10, (4) an antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain amino acid sequence comprises SEQ ID NO:3 and wherein the variable light chain amino acid sequence comprises SEQ ID NO:4, (5) an antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain amino acid sequence comprises SEQ ID NO:7 and wherein the variable light chain amino acid sequence comprises SEQ ID NO:8, or (6) an antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain amino acid sequence comprises SEQ ID NO:11 and wherein the variable light chain amino acid sequence comprises SEQ ID NO:12, wherein the capture molecule is capable of binding the labeled MTX and has less than 6% cross-reactivity with DAMPA (4-Amino-4-deoxy-N-10-methylpteroic acid) in a competitive assay,
and wherein MTX when present in the sample competes with the labeled MTX for binding to the capture molecule; and
detecting an amount of labeled MTX bound to the capture molecule through a signal produced by the labeled MTX, wherein the signal is inversely proportional to the amount of MTX present in the sample.
8. The method of claim 7 , wherein the antibody has less than 1% cross-reactivity with DAMPA (4-Amino-4-deoxy-N-10-methylpteroic acid) in a competitive assay.
9. The method of claim 7 , wherein the antibody has less than 1% cross-reactivity with 7-hydroxy-methotrexate (7-OH-MTX) in a competitive assay.
10. The method of claim 7 , wherein the antibody has less than 1% cross-reactivity with folic acid in a competitive assay.
11. The method of claim 7 , wherein the antibody has less than 1% cross-reactivity with folinic acid in a competitive assay.
12. The method of claim 7 , wherein the antibody has less than 2% cross-reactivity with one or more compounds selected from the group consisting of DAMPA (4-Amino-4-deoxy-N-10-methylpteroic acid), 7-hydroxy-methotrexate (7-OH-MTX), folic acid and folinic acid.